26 research outputs found
Distinct inactive conformations of the dopamine D2 and D3 receptors correspond to different extents of inverse agonism
By analyzing and simulating inactive conformations of the highly-homologous dopamine D2 and D3 receptors (D2R and D3R), we find that eticlopride binds D2R in a pose very similar to that in the D3R/eticlopride structure but incompatible with the D2R/risperidone structure. In addition, risperidone occupies a sub-pocket near the Na+ binding site, whereas eticlopride does not. Based on these findings and our experimental results, we propose that the divergent receptor conformations stabilized by Na+-sensitive eticlopride and Na+-insensitive risperidone correspond to different degrees of inverse agonism. Moreover, our simulations reveal that the extracellular loops are highly dynamic, with spontaneous transitions of extracellular loop 2 from the helical conformation in the D2R/risperidone structure to an extended conformation similar to that in the D3R/eticlopride structure. Our results reveal previously unappreciated diversity and dynamics in the inactive conformations of D2R. These findings are critical for rational drug discovery, as limiting a virtual screen to a single conformation will miss relevant ligands
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High-potency ligands for DREADD imaging and activation in rodents and monkeys.
Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for human applications as well. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine, making it difficult to apply in basic and translational applications. Here we report the development of two novel DREADD agonists, JHU37152 and JHU37160, and the first dedicated 18F positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. We show that JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons, and at their long-range projections, enabling noninvasive and longitudinal neuronal projection mapping
High-potency ligands for DREADD imaging and activation in rodents and monkeys
Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for human applications as well. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine, making it difficult to apply in basic and translational applications. Here we report the development of two novel DREADD agonists, JHU37152 and JHU37160, and the first dedicated 18F positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. We show that JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons, and at their long-range projections, enabling noninvasive and longitudinal neuronal projection mapping
Computational Investigation of Aromatic Oligoamide Foldamers
Foldamers are synthetic oligomers that adopt defined secondary structures in solution. Their functionality relies on their shape. We use all-atom molecular dynamics (MD) simulations with improved force field parameters to study the structure and dynamics of foldamers. This work includes three projects involving aromatic foldamers: 1) DNA-binding foldamers; 2) molecular encapsulation by foldamers; 3) folding-unfolding and handedness inversion in helical foldamers. In the first project, we investigate DNA-binding foldamers. Ligands that are capable of binding DNA in a sequence specific manner and interrupting transcription factor-DNA interactions are of a great interest due their ability to inhibit a number of human cancers. We apply MD to optimize the design of cyclic foldamers (experimentally shown to bind to DNA) by evaluating the influence of the shape of the foldamer on binding affinity and selectivity as well as the dynamics of DNA upon foldamer binding. In the second project, we investigate molecular capsules. Encapsulation can be useful in molecular recognition, catalysis, and drug delivery. Foldamers composed of pyridine and quinoline units have experimentally been shown to form helical capsules and encapsulate small ligands. However, no detailed information on ligand-capsule interactions and dynamics or on the mechanism of encapsulation has been reported, despite the fact that such information is crucial for rational design of capsules. We address these issues through MD simulations. In the third project, we investigate the molecular details of handedness inversion by helical foldamers. As is well known, helical molecules possess handedness, which affects their function. Experimental studies have determined 1 the kinetic rate constants and free energy barriers for racemization for aromatic oligoamides derived from 8-amino-2-quinolinecarboxilic acid. However, the detailed atomistic picture of helix unfolding and handedness inversion is missing. We use MD simulations coupled with energy biasing method, metadynamics, to address this question
Incorporation of Multiple β<sup>2</sup>‑Hydroxy Acids into a Protein <i>In Vivo</i> Using an Orthogonal Aminoacyl-tRNA Synthetase
The programmed synthesis of sequence-defined biomaterials
whose
monomer backbones diverge from those of canonical α-amino acids
represents the next frontier in protein and biomaterial evolution.
Such next-generation molecules provide otherwise nonexistent opportunities
to develop improved biologic therapies, bioremediation tools, and
biodegradable plastic-like materials. One monomer family of particular
interest for biomaterials includes β-hydroxy acids. Many natural
products contain isolated β-hydroxy acid monomers, and polymers
of β-hydroxy acids (β-esters) are found in polyhydroxyalkanoate
(PHA) polyesters under development as bioplastics and drug encapsulation/delivery
systems. Here we report that β2-hydroxy acids possessing
both (R) and (S) absolute configuration
are substrates for pyrrolysyl-tRNA synthetase (PylRS) enzymes in vitro and that (S)-β2-hydroxy acids are substrates in cellulo. Using
the orthogonal MaPylRS/MatRNAPyl synthetase/tRNA pair, in conjunction with wild-type E. coli ribosomes and EF-Tu, we report the cellular
synthesis of model proteins containing two (S)-β2-hydroxy acid residues at internal positions. Metadynamics
simulations provide a rationale for the observed preference for the
(S)-β2-hydroxy acid and provide
mechanistic insights that inform future engineering efforts. As far
as we know, this finding represents the first example of an orthogonal
synthetase that acylates tRNA with a β2-hydroxy acid
substrate and the first example of a protein hetero-oligomer containing
multiple expanded-backbone monomers produced in cellulo
Exploring Substrate Binding in the Extracellular Vestibule of MhsT by Atomistic Simulations and Markov Models
Neurotransmitter:sodium symporters
(NSS) terminate neurotransmission
through Na<sup>+</sup>-driven reuptake of cognate neurotransmitters.
Crystallographically, whereas both substrates and inhibitors have
been found to bind in the central binding (S1) site of NSS, inhibitors
were found to bind to a second binding (S2) site in the extracellular
vestibule (EV) of transporters for leucine (LeuT) and serotonin. On
the basis of computational and experimental studies, we proposed that
substrates bind to the S2 site of LeuT as well and that substrate
binding to the S2 site is essential for Na<sup>+</sup>-coupled symport.
Recent binding experiments show that substrate (l-Trp) binding
in the S2 site of MhsT, another bacterial NSS, is also central to
the allosteric transport mechanism. Here, we used extensive molecular
dynamics simulations combined with Markov state model analysis to
investigate the interaction of l-Trp with the EV of MhsT
and identified potential binding poses of l-Trp as well as
induced conformational changes in the EV. Our computational findings
were validated by experimental mutagenesis studies and shed light
on the ligand binding characteristics of the EV of NSS, which may
facilitate development of allosteric ligands targeting NSS
Exploring Substrate Binding in the Extracellular Vestibule of MhsT by Atomistic Simulations and Markov Models
Neurotransmitter:sodium symporters
(NSS) terminate neurotransmission
through Na<sup>+</sup>-driven reuptake of cognate neurotransmitters.
Crystallographically, whereas both substrates and inhibitors have
been found to bind in the central binding (S1) site of NSS, inhibitors
were found to bind to a second binding (S2) site in the extracellular
vestibule (EV) of transporters for leucine (LeuT) and serotonin. On
the basis of computational and experimental studies, we proposed that
substrates bind to the S2 site of LeuT as well and that substrate
binding to the S2 site is essential for Na<sup>+</sup>-coupled symport.
Recent binding experiments show that substrate (l-Trp) binding
in the S2 site of MhsT, another bacterial NSS, is also central to
the allosteric transport mechanism. Here, we used extensive molecular
dynamics simulations combined with Markov state model analysis to
investigate the interaction of l-Trp with the EV of MhsT
and identified potential binding poses of l-Trp as well as
induced conformational changes in the EV. Our computational findings
were validated by experimental mutagenesis studies and shed light
on the ligand binding characteristics of the EV of NSS, which may
facilitate development of allosteric ligands targeting NSS
The Isomeric Preference of an Atypical Dopamine Transporter Inhibitor Contributes to Its Selection of the Transporter Conformation
Cocaine, a widely
abused psychostimulant, inhibits the dopamine
transporter (DAT) by trapping the protein in an outward-open conformation,
whereas atypical DAT inhibitors such as benztropine have low abuse
liability and prefer less outward-open conformations. Here, we use
a spectrum of computational modeling and simulation approaches to
obtain the underlying molecular mechanism in atomistic detail. Interestingly,
our quantum mechanical calculations and molecular dynamics (MD) simulations
suggest that a benztropine derivative JHW007 prefers a different stereoisomeric
conformation of tropane in binding to DAT compared to that of a cocaine
derivative, CFT. To further investigate the different inhibition mechanisms
of DAT, we carried out MD simulations in combination with Markov state
modeling analysis of wild-type and Y156F DAT in the absence of any
ligand or the presence of CFT or JHW007. Our results indicate that
the Y156F mutation and CFT shift the conformational equilibrium toward
an outward-open conformation, whereas JHW007 prefers an inward-occluded
conformation. Our findings reveal the mechanistic details of DAT inhibition
by JHW007 at the atomistic level, which provide clues for rational
design of atypical inhibitors